Novel high throughput screen reports that benzo(a)pyrene overrides mouse trophoblast stem cell multipotency, inducing SAPK activity, HAND1 and differentiated trophoblast giant cells

• Published in: Placenta (Eastbourne) 2024-07, Vol.152, p.72-85
• Main Authors: Kidder, B.L., Ruden, X., Singh, A., Marben, T.A., Rass, L., Chakravarty, A., Xie, Y., Puscheck, E.E., Awonuga, A.O., Harris, S., Ruden, D.M., Rappolee, D.A.
• Format: Article
• Language: English
• Subjects: Animals; Artificial intelligence; Basic Helix-Loop-Helix Transcription Factors - metabolism; benzo(a)pyrene; Benzo(a)pyrene - pharmacology; Benzo(a)pyrene - toxicity; Cell Differentiation - drug effects; Cells, Cultured; DevTox; Female; Giant Cells - cytology; Giant Cells - drug effects; Giant Cells - metabolism; High throughput screens; High-Throughput Screening Assays - methods; Live imager; Mice; Miscarriage; Pregnancy; SAPK/JNK/MAPK8/9; Stem cells; Stem Cells - drug effects; Stem Cells - metabolism; Stress; Trophoblast stem cells; Trophoblasts - drug effects; Trophoblasts - metabolism
• ISSN: 0143-4004; 1532-3102; 1532-3102
• DOI: 10.1016/j.placenta.2023.12.020

Cultured mouse trophoblast stem cells (mTSC) maintain proliferation/normal stemness (NS) under FGF4, which when removed, causes normal differentiation (ND). Hypoxic, or hyperosmotic stress forces trophoblast giant cells (TGC) differentiate. Hypoxic, hyperosmotic, and genotoxic benzo(a)pyrene (BaP), which is found in tobacco smoke, force down-regulation of inhibitor of differentiation (Id)2, enabling TGC differentiation. Hypoxic and hyperosmotic stress induce TGC by SAPK-dependent HAND1 increase. Here we test whether BaP forces mTSC-to-TGC while inducing SAPK and HAND1. Hand1 and SAPK activity were assayed by immunoblot, mTSC-to-TGC growth and differentiation were assayed at Tfinal after 72hr exposure of BaP, NS, ND, Retinoic acid (RA), or sorbitol. Nuclear-stained cells were micrographed automatically by a live imager, and assayed by ImageJ/FIJI, Biotek Gen 5, AIVIA proprietary artificial intelligence (AI) software or open source, CellPose artificial intelligence/AI software. BaP (0.05-1μM) activated SAPK and HAND1 without diminishing growth. TSC-to-TGC differentiation was assayed with increasingly accuracy for 2–4 N cycling nuclei and >4 N differentiating TGC nuclei, using ImageJ/FIJI, Gen 5, AIVIA, or CellPose AI software. The AIVIA and Cellpose AI software matches human accuracy. The lowest BaP effects on SAPK activation/HAND1 increase are >10-fold more sensitive than similar effects for mESC. RA induces 44–47% 1st lineage TGC differentiation, but the same RA dose induces only 1% 1st lineage mESC differentiation. First, these pilot data suggest that mTSC can be used in high throughput screens (HTS) to predict toxicant exposures that force TGC differentiation. Second, mTSC differentiated more cells than mESC for similar stress exposures, Third, open source AI can replace human micrograph quantitation and enable a miscarriage-predicting HTS. •Benzo(a)pyrene activates SAPK and HAND1, which is necessary for TGC differentiation.•Benzo(a)pyrene overrides TSC stemness and forces TGC differentiation.•A new TSC to TGC high throughput screen was made using TSC assayed in a live imager.•AIVIA AI or open source CellPose produced high quality TSC-to-TGC size and number analysis.•enzo(a)pyrene and retinoic acid cause mTSC >>> mESC differentiation, suggesting miscarriage.